LOCUS       sequence-304865-        3766 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mutisite Gateway mediated vector construction.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3766)
  AUTHORS   Yoshimatsu S, Sone T, Nakajima M, Sato T, Okochi R, Ishikawa M, 
            Nakamura M, Sasaki E, Shiozawa S, Okano H
  TITLE     A versatile toolbox for knock-in gene targeting based on the 
            Multisite Gateway technology.
  JOURNAL   PLoS One. 2019 Aug 27;14(8):e0221164. doi: 
            10.1371/journal.pone.0221164. eCollection 2019.
  PUBMED    31454364
REFERENCE   2  (bases 1 to 3766)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "PLoS One.";
            date: "2019-08-27"; volume: "14(8):e0221164. doi"; pages: " 
            10.1371/journal.pone.0221164. eCollection 2019"
COMMENT     Sequence Label: pENTR-L1-PGK-Neo-L2
FEATURES             Location/Qualifiers
     source          1..3766
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             58..867
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(125..144)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      1010..1598
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1499..1518
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1733..1760)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1852..1938)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    1991..2090
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        2097..2596
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2132..2155)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2529..2548
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     promoter        2613..2660
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             2679..3482
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2742..2761)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3352..3371
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     protein_bind    complement(3602..3701)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
ORIGIN
        1 atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag gggtgttatg
       61 agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga tgctgattta
      121 tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat ctatcgcttg
      181 tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag cgttgccaat
      241 gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc tcttccgacc
      301 atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc gatccccgga
      361 aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat tgttgatgcg
      421 ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc ttttaacagc
      481 gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt ggttgatgcg
      541 agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa agaaatgcat
      601 aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc acttgataac
      661 cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt cggaatcgca
      721 gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc tccttcatta
      781 cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa attgcagttt
      841 catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta acactggcag
      901 agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct taacgtgagt
      961 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     1021 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     1081 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     1141 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     1201 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     1261 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     1321 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     1381 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     1441 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     1501 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     1561 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     1621 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     1681 attctgtgga taaccgtatt accgctagcc aggaagagtt tgtagaaacg caaaaaggcc
     1741 atccgtcagg atggccttct gcttagtttg atgcctggca gtttatggcg ggcgtcctgc
     1801 ccgccaccct ccgggccgtt gcttcacaac gttcaaatcc gctcccggcg gatttgtcct
     1861 actcaggaga gcgttcaccg acaaacaaca gataaaacga aaggcccagt cttccgactg
     1921 agcctttcgt tttatttgat gcctggcagt tccctactct cgcgttaacg ctagcatgga
     1981 tctcgggccc caaataatga ttttattttg actgatagtg acctgttcgt tgcaacaaat
     2041 tgatgagcaa tgctttttta taatgccaac tttgtacaaa aaagcagaat tctaccgggt
     2101 aggggaggcg cttttcccaa ggcagtctgg agcatgcgct ttagcagccc cgctgggcac
     2161 ttggcgctac acaagtggcc tctggcctcg cacacattcc acatccaccg gtaggcgcca
     2221 accggctccg ttctttggtg gccccgtcgc gccaccttct actcctcccc tagtcaggaa
     2281 gttccccccc gccccgcagc tcgcgtcgtg caggacgtga caaatggaag tagcacgtct
     2341 cactagtctc gtgcagatgg acagcaccgc tgagcaatgg aagcgggtag gcctttgggg
     2401 cagcggccaa tagcagcttt gctccttcgc tttctgggct cagaggctgg gaaggggtgg
     2461 gtccgggggc gggctcaggg gcgggctcag gggcggggcg ggcgcccgaa ggtcctccgg
     2521 aggcccggca ttctgcacgc ttcaaaagcg cacgtctgcc gcgctgttct cctcttcctc
     2581 atctccgggc ctttcgacct gcagcagcac gtgttgacaa ttaatcatcg gcatagtata
     2641 tcggcatagt ataatacgac aaggtgagga actaaaccat gggatcggcc attgaacaag
     2701 atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg
     2761 cacaacagac gatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc
     2821 cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag
     2881 cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca
     2941 ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat
     3001 ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata
     3061 cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac
     3121 gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc
     3181 tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg
     3241 tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg
     3301 gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta
     3361 cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg
     3421 gtatcgccgc ccccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct
     3481 gagcgggact ctggggttcg aataaagacc gaccaagcga cgtctgagag ctccctggcg
     3541 aattcggtac caataaaaga gctttatttt catgatctgt gtgttggttt ttgtgtgcgg
     3601 cgcgcagctt tcttgtacaa agttggcatt ataagaaagc attgcttatc aatttgttgc
     3661 aacgaacagg tcactatcag tcaaaataaa atcattattt gccatccagc tgcagctctg
     3721 gcccgtgtct caaaatctct gatgttacat tgcacaagat aaaaat
//
